CpG islands which are generally not methylated in normal cells, frequently become de novo methylated in cancer and this epigenetic modification may contribute to the silencing of tumor suppressor and DNA repair genes. The true extent of these epigenetic changes and the mechanisms underlying the abnormal methylation of CpG islands are almost completely unknown. This proposal seeks to use a new genome scanning method Methylation Sensitive Arbitrarily Primed-PCR (MS-AP-PCR) to detect abnormally methylated CpG islands and to determine how patterns of methylation at unselected sites change during the progression of human bladder cancers. The MS-AP-PCR method allows for the cloning, sequencing and identification of methylated fragments and will be coupled with a quantitative assay (Ms-SNuPE) so that the evolution of methylation changes can be accurately measured. In the first specific aim Dr. Jones will continue work in progress to sequence and identify additional CpG islands which become methylated in invasive bladder cancers when compared to adjacent apparently normal uroepithelium. Specific Aim 2 will test the hypothesis that methylation changes are already present in this apparently normal urothelium and may be associated with a "field defect" in the epithelium. Specific Aims 3 and 4 will contrast methylation patterns in low-grade papillary (Ta) tumors with invasive cancers and determine whether additional changes occur during metastasis. Specific Aim 5 will use the in vitro culture of bladder tumors to test the hypothesis that methylation of CpG island within the promoters of the p16 tumor suppressor gene is selected from a background of functionally irrelevant methylation changes to give rise to immortal cells. The investigators believe that the application of the non-directed MS-AP-PCR approach to a well understood tumor such as bladder cancer at different stages of progression will provide answers to some of the key questions in this field.